Sensory ASIC3 channel exacerbates psoriatic inflammation via a neurogenic pathway in female mice

Psoriasis is an immune-mediated skin disease associated with neurogenic inflammation, but the underlying molecular mechanism remains unclear. We demonstrate here that acid-sensing ion channel 3 (ASIC3) exacerbates psoriatic inflammation through a sensory neurogenic pathway. Global or nociceptor-specific Asic3 knockout (KO) in female mice alleviates imiquimod-induced psoriatic acanthosis and type 17 inflammation to the same extent as nociceptor ablation. However, ASIC3 is dispensable for IL-23-induced psoriatic inflammation that bypasses the need for nociceptors. Mechanistically, ASIC3 activation induces the activity-dependent release of calcitonin gene-related peptide (CGRP) from sensory neurons to promote neurogenic inflammation. Botulinum neurotoxin A and CGRP antagonists prevent sensory neuron-mediated exacerbation of psoriatic inflammation to similar extents as Asic3 KO. In contrast, replenishing CGRP in the skin of Asic3 KO mice restores the inflammatory response. These findings establish sensory ASIC3 as a critical constituent in psoriatic inflammation, and a promising target for neurogenic inflammation management.

1.The novelty of this study is limited without knowing the ASIC3-expressing neuron types and how ASIC3 of skin afferents is activated in the context of IMQ-induced inflammation.Also, how CGRP signaling can lead to abnormal epidermal proliferation and up-regulation of type 17 cytokines should be addressed.2. In general, the statistical analyses are not appropriate.In most 2x2 experimental groups, 2-way ANOVA instead of one-way ANOVA should be used.3. Baseline data (vehicle control) of epidermal thickness, IL-17, IL-22, and IL-23 are missing in the Figure 1. 4. In Figure 2a, the validation of Nav1.8 conditional ASIC3 knockout should be analyzed by a quantitative approach (e.g., QPCR). 5. How are ASIC3 and TRPV1 co-localized in skin afferents innervating dorsal skin? 6.Is the IL-23-induced psoriatic inflammation (on ears) a relevant model with IMQ-induced one (on dorsal skin)?The rationale of the IL-23-induced psoriatic inflammation experiment is not clear.It may be more important to know whether IL-23 is a downstream signaling of ASIC3 activation and/or CGRP receptors.7. The Figure 3b does not support that IL-23 treatment can induce splenomegaly.8.It is surprising to see KCL-induced CGRP release is ASIC3-dependent in cultured DRG neurons (Fig. 5a).Of note, GMQ also induced CRGP release in ASIC3 knockout.What are the interpretations of these data?9.The rationale to measure CGRP in the skin explant is not clear.The different CGRP levels between normal and IMQ-treated skin cannot tell the contribution of neurogenic effect from skin afferents.10.The data of Figure 5c is not convincing.Quantitative analysis is needed.11.It is not clear to what extent the ASIC3-expressing skin afferents could contribute to the IMQinduced psoriatic inflammation.12.The discussion section should be more carefully organized.There is too much speculation in the discussion.13.Were all studied performed in male or female mice (or both)?What are the mouse ages?14.What were the efficacies of ASIC3 knockdown and gene rescue in the supplementary Figure 2? 15.How were the skin samples collected and processed for ELISA assays?Especially, it is important to know how the lesional skin is defined.
Reviewer #2 (Remarks to the Author): The current study highlights the importance of ASIC3, an ion channel previously overlooked in skin inflammation but known for its involvement in itch and nociception.The investigators found that ASIC3 contributes to the psoriatic skin phenotype and accompanying type 17 inflammation.Knocking out Asic3 globally or specifically in nociceptors in mice significantly reduced psoriatic acanthosis and type 17 inflammation induced by IMQ, comparable to nociceptor ablation.However, ASIC3 was not essential for IL-23-induced psoriatic inflammation, which can bypass the need for nociceptors.Further investigation revealed that ASIC3 activation leads to activity-dependent release of CGRP from sensory neurons.By using botulinum neurotoxin A and CGRP antagonists, they were able to prevent the sensory neuron-mediated exacerbation of psoriatic inflammation, similar to the effects observed with Asic3 knockout.Conversely, replenishing CGRP in Asic3 knockout skin restored the inflammatory response.The study establishes sensory ASIC3 as a critical component in psoriatic inflammation and a promising target for managing chronic skin diseases.While the study is potentially interesting, there are several results that do not support the conclusion they have made.Additionally, there are several important experiments that are missing.
From the experiment shown in Figure 3b they claim IL-23 administration induced significant splenomegaly in both groups.However, the experiment does not have any vehicle control.As compared to the vehicle controls in Figure 1b (~70 mg), it does not have significant increase in spleen weight after IL-23 intradermal administration in both WT and KO in Figure 3b (also around 70 mg).
Are there any significant increases in Ki67 positive cells in WT and KO mice after IL-23 treatment as compared to PBS controls (Figure 3d)?If not, it suggests IL-23 treatment does not induce significant cell proliferation.In this study, they need to specify the type of cells that Ki67 positive cells represent.
It is unclear whether BoNT/A can directly block CGRP release which is an important point for the study.
Another important point remains unaddressed is what activate ASCI3 in psoriatic skin.Can they detect acidification or LPC increase in psoriatic skin?
In Figure 5a,b, GMQ seems also increase CGRP release in ASIC3 KO DRG.If this is the case, it suggests AISC3 is not the only mediator to induce CGRP release.
The quality of nerve image in Fig 5C is poor.It is hard to see any nerve staining.They should include negative control for CGRP staining.Also, they need to quantify the nerve counts in order to make the conclusion.CGRP injection increased only IL17 but not other cytokines.So CGRP itself does not induce a full-scale inflammatory responses. 1

Reviewer #1:
This is an interesting study to show a role of sensory neuron ASIC3 in psoriatic inflammation in mouse models.The  We thank the reviewer for the positive comments.and 9, and their legends, page 14, the last paragraph to page 15, the 2 nd paragraph).
2. In general, the statistical analyses are not appropriate.In most 2x2 experimental groups, 2-way ANOVA instead of one-way ANOVA should be used.1.

Baseline data (vehicle control) of epidermal thickness, IL-17, IL-22, and IL-23 are missing in the Figure
Included as suggested (please see new Fig. 1).2a, the validation of Nav1.8 conditional ASIC3 knockout should be analyzed by a quantitative approach (e.g., QPCR).Done accordingly (please see new Supplementary Fig. 2).

How are ASIC3 and TRPV1 co-localized in skin afferents innervating dorsal skin?
Done accordingly in an ASIC3-myc reporter mice (please see Supplementary Fig. 5 and its legend, page 9, end of the last paragraph to page 10, beginning of the 1 st paragraph).

Is the IL-23-induced psoriatic inflammation (on ears) a relevant model with IMQinduced one (on dorsal skin)?
The rationale of the IL-23-induced psoriatic inflammation experiment is not clear.It may be more important to know whether IL-23 is a downstream signaling of ASIC3 activation and/or CGRP receptors.

It is surprising to see KCL-induced CGRP release is ASIC3-dependent in cultured
DRG neurons (Fig. 5a).Of note, GMQ also induced CRGP release in ASIC3 knockout.

What are the interpretations of these data?
We appreciate the reviewer for bringing this to our attention.After a thorough reassessment of the experiments, we identified an error in the previous version of our manuscript regarding the interpretation of the KCl-induced CGRP release in DRG neurons.We have rectified this in the revised manuscript, clarifying that high potassium (30 mM KCl) induces a significant increase in CGRP secretion from both Asic3 +/+ and Asic3 -/-DRG neurons (please see new Fig.5b).5c is not convincing.Quantitative analysis is needed.

It is not clear to what extent the ASIC3-expressing skin afferents could contribute to the IMQ-induced psoriatic inflammation.
We appreciate the reviewer's point regarding the necessity to clarify the extent by which ASIC3-expressing skin afferents contribute to IMQ-induced psoriatic inflammation.Our results demonstrate that Asic3 KO mice exhibit a comparable improvement in psoriatic inflammation as those with their TRPV1 + nociceptors ablated.This suggests that ASIC3-expressing skin afferents play a role comparable to nociceptive sensory neurons in driving psoriatic inflammation.
12. The discussion section should be more carefully organized.There is too much speculation in the discussion.
In response to the reviewer's valuable feedback, we have reorganized and refined the Discussion section to enhance clarity and reduce speculative elements.We have also incorporated insights gained from our new experiments (please see new Discussion section).

Were all studied performed in male or female mice (or both)? What are the mouse ages?
The information is now provided in the revised manuscript (please see page 22, middle of the 1 st paragraph).

How were the skin samples collected and processed for ELISA assays? Especially, it is important to know how the lesional skin is defined.
Six hours after the final IMQ treatment, we harvested a 12 mm punch biopsy from the treated skin, which was then placed in a 24-well plate containing 1 ml DMEM.

Reviewer #2:
The current study highlights the importance of ASIC3, an ion channel previously overlooked in skin inflammation but known for its involvement in itch and nociception.

The investigators found that ASIC3 contributes to the psoriatic skin phenotype and accompanying type 17 inflammation. Knocking out Asic3 globally or specifically in nociceptors in mice significantly reduced psoriatic acanthosis and type 17 inflammation induced by IMQ, comparable to nociceptor ablation. However, ASIC3
was not essential for IL-23-induced psoriatic inflammation, which can bypass the need for nociceptors.Further investigation revealed that ASIC3 activation leads to activitydependent release of CGRP from sensory neurons.By using botulinum neurotoxin A and CGRP antagonists, they were able to prevent the sensory neuron-mediated exacerbation of psoriatic inflammation, similar to the effects observed with Asic3 knockout.Conversely, replenishing CGRP in Asic3 knockout skin restored the inflammatory response.The study establishes sensory ASIC3 as a critical component in psoriatic inflammation and a promising target for managing chronic skin diseases.
While the study is potentially interesting, there are several results that do not support the conclusion they have made.Additionally, there are several important experiments that are missing.
We appreciate the positive feedback from the reviewer.Additionally, following the reviewer's suggestions, we conducted additional experiments.

Are there any significant increases in Ki67 positive cells in WT and KO mice after
IL-23 treatment as compared to PBS controls (Figure 3d)?If not, it suggests IL-23 treatment does not induce significant cell proliferation.In this study, they need to specify the type of cells that Ki67 positive cells represent.

paragraph to page 16, the 1 st paragraph). These insights shed lights on potential mechanisms of ASIC3 activation within the context of IMQ-induced inflammation.
5. In Figure 5a,b, GMQ seems also increase CGRP release in ASIC3 KO DRG.If this is the case, it suggests AISC3 is not the only mediator to induce CGRP release.
Regarding the observation that GMQ induced CGRP release in Asic3 knockout mice, it is essential to note that GMQ has multiple molecular targets, including acting as a nonproton agonist of ASIC3 and as an antagonist of GABA A receptors.
The effects of GMQ on ASIC3 knockout could be attributed to its influence on GABA A receptors.Supporting this view, another GABA A receptor antagonist, bicuculline, also induced CGRP release in an ASIC3-independent manner (please see new Supplementary Fig. 7b and its legend, page 13, middle of the 1 st paragraph).

The quality of nerve image in Fig 5C is poor. It is hard to see any nerve staining.
They should include negative control for CGRP staining.Also, they need to quantify the nerve counts in order to make the conclusion.CGRP injection increased only IL17 but not other cytokines.So CGRP itself does not induce a full-scale inflammatory responses.
We apologize for the previous image quality issue and appreciate the reviewer's feedback.To address this concern, we have conducted additional immunofluorescence staining for both CGRP and PGP9.5 and obtained nerve images with enhanced quality.Furthermore, we have quantified nerve counts to provide a more comprehensive analysis (please see Fig. 5d,e and its legend).
Regarding the impact of CGRP injection on cytokine release, we reevaluated our data.By directly comparing the differences between the vehicle and CGRPtreated groups in Asic3 -/-mice using Student's t-test, we found that CGRP injection increased IL-17, IL-22, and IL-23 cytokines (please see new Fig.6e and its legend).This suggests that CGRP itself induces a full-scale inflammatory response.9. Is LPC16:0 detectable in skin samples?Yes, LPC16:0 was detected in skin samples, but its level was not significantly higher in IMQ-treated mice than vehicle-treated ones.Furthermore, LPC16:0 did not induce CGRP release at neutral pH in Asic3 +/+ DRG neurons (please see new Supplementary Fig. 12a,d).

Issues of two-way ANOVA in
Minor points: 1.In page 9, line 179.The sentence is confusing.The data were compared between conditional rescue of ASIC3 in Nav1.8-Cre mice and ASIC3 knockdown mice (but not WT mice).
Correct accordingly (please see Page 9, the 2 nd paragraph).

In page 22, line 461. Is it exon 11? Or exon 1?
Correct accordingly (please see the Supplementary Fig. 5 and its legend).Correct accordingly (please see the Methods section).

The dose of Bicuculine is missing in the Methods (and Figure legends).
However, although much effort has proved a role for ASIC3 involved in IMQ-induced psoriatic inflammation, no detail mechanistic insight had offered to advance our understanding how the ASIC3-mediated neurogenic effects are activated and how this ASIC3 signaling is different from the TRPV1 pathway in the context of IMQ-induced psoriatic inflammation.In response to the insightful feedback from the reviewer, we have performed additional experiments, incorporated new data, and provided expanded explanations in the Discussion section.Specifically, we have addressed the mechanisms triggering ASIC3-mediated neurogenic effects (please see Fig. 7, Supplementary Figs. 10 and 11, and their legends, page 15, the last paragraph to page 16, the 1 st paragraph) and elucidated the distinctions between ASIC3 signaling and the TRPV1 pathway within the context of IMQ-induced psoriatic inflammation (please see Supplementary Fig. 5 and its legend, page 9, end of the last paragraph to page 10, beginning of the 1 st paragraph).Beneath are some specific comments.1.The novelty of this study is limited without knowing the ASIC3-expressing neuron types and how ASIC3 of skin afferents is activated in the context of IMQ-induced inflammation.Also, how CGRP signaling can lead to abnormal epidermal proliferation and up-regulation of type 17 cytokines should be addressed.In response to the reviewer's suggestion, we have integrated new data obtained from a recently established ASIC3-myc reporter mouse model.This includes a comprehensive characterization of ASIC3 distribution specifically in TRPV1 + neurons and skin afferents (please see Supplementary Fig. 5 and its legend, page 9, end of the last paragraph to page 10, beginning of the 1 st paragraph).Furthermore, we have included novel findings showing skin acidosis and increased presence of LPC in IMQ-induced psoriatic lesions (please see Fig. 7, Supplementary Figs. 10 and 11, and their legends, page 15, the last paragraph to page 16, the 1 st paragraph).These insights shed light on potential mechanisms of ASIC3 activation within the context of IMQ-induced inflammation.In addition, we have conducted additional experiments to specifically address how CGRP signaling is involved in abnormal epidermal proliferation and upregulation of type 17 cytokines (please see Fig. 7, Supplementary Figs. 8 the IMQ-induced model (on dorsal skin).We have clarified the rationale for the IL-23-induced psoriatic inflammation experiment (please see page 11, beginning of the 1st paragraph).In response to the reviewer's suggestion, we have conducted additional experiments utilizing a co-culture system involving DRG neurons and bone marrow-derived DCs (BMDCs).These experiments demonstrate that IL-23 is indeed a downstream signaling molecule produced in response to acid stimulation of ASIC3 activation and the subsequent activation of CGRP receptors (please see new Supplementary Fig. 8, page 14, the last paragraph to page 15, the 1 st paragraph).7. The Figure 3b does not support that IL-23 treatment can induce splenomegaly.Yes.The lack of splenomegaly observed after local IL-23 injection in both groups suggests the absence of systemic effects (please see new Fig.3b, page 11, middle of the 1st paragraph).
Asic3 knockout mice, it is essential to note that GMQ has multiple molecular targets, including acting as a nonproton agonist for ASIC3 (Ref. 1) and as an antagonist for GABA A receptors (Ref.2).The effects of GMQ in ASIC3 knockout are likely attributed to its influence on GABA A receptors.Consistently, another GABA A receptor antagonist, bicuculline also induced CGRP release in an ASIC3independent manner (please see new Supplementary Fig. 7b and its legend, page 13, middle of the 1 st paragraph).References: 1. Yu Y, Chen Z, Li WG, Cao H, Feng EG, Yu F, Liu H, Jiang H, Xu TL.A nonproton ligand sensor in the acid-sensing ion channel.Neuron 2010; 68(1): 61-72.2. Xiao X, Zhu MX, Xu TL. 2-Guanidine-4-methylquinazoline acts as a novel competitive antagonist of A type γ-aminobutyric acid receptors.Neuropharmacology 2013; 75: 126-37.9.The rationale to measure CGRP in the skin explant is not clear.The different CGRP levels between normal and IMQ-treated skin cannot tell the contribution of neurogenic effect from skin afferents.The primary reason for assessing CGRP levels in the skin explant was to investigate local CGRP release in the context of IMQ-induced psoriatic inflammation.We sought to identify differences in CGRP levels between normal and IMQ-treated skin, with an intention to establish a potential contribution of neurogenic effects from skin afferents to the observed differences.We acknowledge that these data alone do not definitively pinpoint to the specific origin of CGRP in the skin explant.However, in conjunction with other complementary approaches, including the assessment of CGRP levels in cultured DRG neurons and the measurement of CGRP + and PGP9.5 + nerve densities in psoriatic lesions, the observed changes in CGRP levels in the IMQ-treated skin compared to the normal skin explant align with the influence of neurogenic effects from skin afferents.This reinforces the proposition that neurogenic CGRP is a pivotal driver of psoriatic inflammation.
The samples were incubated in a 32 °C shaking incubator (150 rpm) for 45 min, and the media were collected for analysis using the CGRP EIA kit (Cayman Chemical Co.) following the procedures outlined in the literature 1 (please see the section of CGRP release assay from skin explants in Methods).It is crucial tonote that the punch biopsies were obtained from either non-lesioned or lesioned skin, where the sample corresponds to the area of the dorsal skin treated with either vehicle or IMQ, as confirmed by H & E staining.No lesioned skin areas were observed in vehicle-treated samples.References: 1. Cohen JA, Edwards TN, Liu AW, Hirai T, Jones MR, Wu J, Li Y, Zhang S, Ho J, Davis BM, Albers KM, Kaplan DH.Cutaneous TRPV1 + neurons trigger protective innate type 17 anticipatory immunity.Cell.2019; 178(4): 919-932.e14.
Figure 3b they claim IL-23 administration induced significant splenomegaly in both groups.However, the experiment does not have any vehicle control.As compared to the vehicle controls in Figure 1b (~70 mg), it does not have significant increase in spleen weight after IL-23 intradermal administration in both WT and KO in Figure 3b (also around 70 mg).We apologize for any confusion caused by the previous version of our manuscript.In response to the reviewer's comment, we administered IL-23 (1 μg) by injection into one ear every other day for a total of 8 days.The results indicate that the IL-23 injection did not induce splenomegaly in either Asic3 +/+ or Asic3 -/-mice (compared to the vehicle control groups, please see new Fig.3b and its legend, page 11, middle of the 1 st paragraph).
Figure 3b (and other Figures) are identified.What is the F value? Done accordingly (please see the Figure legends).
issue is concerned.The Nav1.8-Cre mice were generated by Dr. Rohini Kuner's group.It is not appropriate to use the mice from Dr. Dr. Xu Zhang without acknowledgement of the original authors.